The aim of these studies is to elucidate structural and dynamic mechanisms of molecular machines composed of phosphoinositide/protein complexes as they function in modulating membrane processes. We focus on insulin-mediated GLUT4 glucose transporter translocation to the plasma membrane of adipocytes as our model system, a seminal problem in biology that merges the fields of cell signaling and membrane trafficking. Application of unique TIRF technology already developed within this Program Project, and new technology to be developed in Project 4, positions us to answer previously intractable questions in this field. We propose to define trajectories and dynamics of GLUT4-containing vesicles within about 200nm of the plasma membrane (TIRF zone), using novel methods that enable localization of particles at resolutions of 50nm or less. New data obtained since the last submission shows we can now identify and quantify docking and fusion events during insulin-mediated exocytosis of these vesicles in the TIRF zone. Thus, we can now address the central hypothesis that insulin regulates the kinetics of GLUT4- containing vesicle docking/fusion processes through modulating vesicles associated with either of 2 specific functional phosphoinositide-based protein complexes. We hypothesize that one subset of GLUT4-containing exocytic vesicles rapidly recycle and fuse through a mechanism that depends upon PI(3)P/Rabenosyn-5/EHD1/EHBP1/Rab5/Rab4 complexes. We hypothesize that a second subset of GLUT4- containing exocytic vesicles more slowly recycle and fuse through a mechanism that depends upon PI(3,4,5)P3/FIP2/EHD1/EHBP1/Rab11 complexes. All our efforts are now directed to testing these focused hypotheses related to the nature of GLUT4-containing exocytic vesicles that proceed to docking and fusion with the plasma membrane. We propose to define the 3D paths that GLUT4-containing vesicles traverse during recycling and exocytosis relative to other cargo(EGFR and transferrin receptor with Project 2). Using high resolution TIRF microscopy with exofacially labeled myc-GLUT4-EGFP, we propose to track trajectories of insulin-sensitive, GLUT4-containing vesicles to test whether GLLJT4 in Rab5-positive early endosomes rapidly recycles to fuse with the plasma membrane in a "short circuit" pathway. The combination of unique imaging tools with biochemical approaches provides a powerful way to dissect out the functions of phosphoinositide-based complexes containing EHD1 and Rab proteins in GLUT4 dynamics. We shall also test whether complexes of PI(3,4,5)P3/Akt2/substrates function in docking/fusion of GLUT4-containing vesicles and regulate specific steps in these pathways in the TIRF zone. With Project 3, we will also define the membrane topographies and structures of molecular complexes that act on GLUT4-containing vesicles in the TIRF zone, and elucidate the mechanisms involved in their actions.